Cdt1 Self-associates via the Winged-Helix Domain of the Central Region during the Licensing Reaction, Which Is Inhibited by Geminin.

The initiation of DNA replication is tightly controlled by the licensing system that loads replicative DNA helicases onto replication origins to form pre-replicative complexes (pre-RCs) once per cell cycle. Cdc10-dependent transcript 1 (Cdt1) plays an essential role in the licensing reaction by recruiting mini-chromosome maintenance (MCM) complexes, which are eukaryotic replicative DNA helicases, to their origins via direct protein-protein interactions. Cdt1 interacts with other pre-RC components, the origin recognition complex, and the cell division cycle 6 (Cdc6) protein; however, the molecular mechanism by which Cdt1 functions in the MCM complex loading process has not been fully elucidated. Here, we analyzed the protein-protein interactions of recombinant Cdt1 and observed that Cdt1 self-associates via the central region of the molecule, which is inhibited by the endogenous licensing inhibitor, geminin. Mutation of two ß-strands of the winged-helix domain in the central region of Cdt1 attenuated its self-association but could still interact with other pre-RC components and DNA similarly to wild-type Cdt1. Moreover, the Cdt1 mutant showed decreased licensing activity in Xenopus egg extracts. Together, these results suggest that the self-association of Cdt1 is crucial for licensing.

Biphasic Increases of Cell Surface Calreticulin Following Treatment with Mitoxantrone.

Calreticulin (CRT) and calnexin (CNX), homologous major chaperones in the endoplasmic reticulum (ER), are known to translocate to the cell surface in response to chemotherapeutic agents, such as mitoxantrone (MIT), and cellular stresses, including apoptosis. Cell surface CRT (ecto-CRT) is relevant to the phagocytic uptake of cancer cells and dying cells, and pre-apoptotic exposure of CRT has been reported to result in enhanced immunogenicity of dying tumor cells, serving as a damage-associated molecular pattern (DAMP). In this study, HT-29 cells were treated with MIT to induce ER stress, and ecto-CRT and cell surface CNX were quantified by flow cytometry in the absence or presence of caspase inhibitors, a calpain inhibitor, or a scavenger of reactive oxygen species. The biphasic (early transient and late sustained) increase of ecto-CRT on HT-29 cells was observed after treatment with MIT. We confirmed that the early increase in ecto-CRT after 4 h of MIT treatment was not related to apoptosis, whereas the increase of ecto-CRT, as well as that of cell-surface CNX, during the later stage of treatment was caspase dependent and related to apoptosis. In addition, our results suggested that the early peak of ecto-CRT was mediated by activation of caspase 8 by ER stress. Thus, the physiological significance of the late increases in cell-surface CRT and/or CNX might be considered an "eat-me signal" for the removal of dead cells by phagocytosis, while the early increase in ecto-CRT caused by ER stress might enhance the immunogenicity of stressed tumor cells.

Mutant analysis of Cdt1's function in suppressing nascent strand elongation during DNA replication in Xenopus egg extracts.

The initiation of DNA replication is strictly regulated by multiple mechanisms to ensure precise duplication of chromosomes. In higher eukaryotes, activity of the Cdt1 protein is temporally regulated during the cell cycle, and deregulation of Cdt1 induces DNA re-replication. In previous studies, we showed that excess Cdt1 inhibits DNA replication by suppressing progression of replication forks in Xenopus egg extracts. Here, we investigated the functional regions of Cdt1 that are required for the inhibition of DNA replication. We constructed a series of N-terminally or C-terminally deleted mutants of Cdt1 and examined their inhibitory effects on DNA replication in Xenopus egg extracts. Our results showed that the region spanning amino acids (a. a.) 255-620 is required for efficient inhibition of DNA replication, and that, within this region, a. a. 255-289 have a critical role in inhibition. Moreover, one of the Cdt1 mutants, Cdt1 R285A, was compromised with respect to the licensing activity but still inhibited DNA replication. This result suggests that Cdt1 has an unforeseen function in the negative regulation of DNA replication, and that this function is located within a molecular region that is distinct from those required for the licensing activity.

Anti-proliferative effect of Fe(III) complexed with 1-(2-hydroxy-3-methoxybenzaldehyde)-4-aminosalicylhydrazone in HepG2 cells.

We previously developed a chelating ligand, 1-(2-hydroxy-3-methoxybenzaldehyde)-4-aminosalicylhydrazone (HMB-ASH), which can chelate Fe(III) to form a complex. The HMB-ASH-Fe(III) complex exhibits a dose-dependent anti-proliferative effect in HepG2 cells, whereas the ligand, HMB-ASH, and Fe(III) alone had no considerable effect. The HMB-ASH-Fe(III) complex was composed of Fe(III):HMB-ASH (1:2), as determined by high-performance liquid chromatography with high-resolution mass spectrometry. The IC50 value was approximately 20 µM, which was comparable to those of the anti-cancer drugs oxaliplatin (OXP) and etoposide (ETP) under the same conditions. Similar to OXP and ETP, HMB-ASH-Fe(III) induced apoptosis in HepG2 cells, as revealed by terminal deoxynucleotidyl transferase fluorescein-12-dUTP nick end labeling assay.

Transcriptional regulation of fucosyltransferase 1 gene expression in colon cancer cells.

The α 1,2-fucosyltransferase I (FUT1) enzyme is important for the biosynthesis of H antigens, Lewis B, and Lewis Y. In this study, we clarified the transcriptional regulation of FUT1 in the DLD-1 colon cancer cell line, which has high expression of Lewis B and Lewis Y antigens, expresses the FUT1 gene, and shows α 1,2-fucosyltransferase (FUT) activity. 5'-rapid amplification of cDNA ends revealed a FUT1 transcriptional start site -10 nucleotides upstream of the site registered at NM_000148 in the DataBase of Human Transcription Start Sites (DBTSS). Using the dual luciferase assay, FUT1 gene expression was shown to be regulated at the region -91 to -81 nt to the transcriptional start site, which contains the Elk-1 binding site. Site-directed mutagenesis of this region revealed the Elk-1 binding site to be essential for FUT1 transcription. Furthermore, transfection of the dominant negative Elk-1 gene, and the chromatin immunoprecipitation (CHIp) assay, supported Elk-1-dependent transcriptional regulation of FUT1 gene expression in DLD-1 cells. These results suggest that a defined region in the 5'-flanking region of FUT1 is critical for FUT1 transcription and that constitutive gene expression of FUT1 is regulated by Elk-1 in DLD-1 cells.

Unlike natural killer (NK) p30, natural cytotoxicity receptor NKp44 binds to multimeric α2,3-NeuNAc-containing N-glycans.

Natural cytotoxicity receptor 2 (NCR2 or natural killer (NK)p44) and NCR3 (NKp30) bind to heparin and heparin sulfate; however, other natural ligands have yet to be identified. We previously reported that NCR1 (NKp46) can bind to multimeric NeuNAc-containing N-glycans and sulfated glycans. In this study, we investigated whether NKp44 and NKp30 can bind to NeuNAc-containing glycans using their common recombinant extracellular domain tagged with 6×His (NKp44-H6 and NKp30-H6). NKp44-H6, but not NKp30-H6, bound multimeric sialyl Lewis X expressing transferrin secreted by HepG2 cells (HepTF) with a K(d) of 420 nM. Competitive and direct binding assays revealed that NKp44-H6 mainly recognizes α2,3-NeuNAc residues on non-reducing ends of N-glycans on HepTF. Moreover, site-directed mutants of NKp44-H6, such as R47Q, R55Q, R92Q, R95Q, K103Q, and R106Q, had reduced binding to α2,3-sialylated N-glycans. These results suggest that NKp44 binds to α2,3-sialylated N-glycans through ionic interactions, and that these binding sites might have some overlap with heparin binding sites.

Distinct roles in phagocytosis of the early and late increases of cell surface calreticulin induced by oxaliplatin.

Calreticulin (CRT), a chaperone typically located in the endoplasmic reticulum (ER), is known to translocate to the cell surface in response to anticancer drugs. Cell surface CRT (ecto-CRT) on apoptotic or pre-apoptotic cells serves as an "eat me" signal that can promote phagocytosis. In this study, we observed the biphasic (early transient and late sustained) increase of ecto-CRT on HT-29 cells after treatment with oxaliplatin (L-OHP). To investigate the role of ecto-CRT that accumulates in the early and late phases as "eat me" signals, we examined the phagocytosis of HT-29 cells by macrophage-like cells and dendritic cell (DC) -like cells prepared from THP-1 cells. The results indicated that the early ecto-CRT-expressed cells were phagocytosed by immature DC-like cells, and the late ecto-CRT-expressed cells were phagocytosed primarily by macrophage-like cells, while mature DC-like cells did not respond to the either class of ecto-CRT-expressed cells. Both types of phagocytotic events were inhibited by CRT Blocking Peptide, suggesting that such events depended on the ecto-CRT. Our results suggested that the early increase of ecto-CRT is related to phagocytosis as part of immunogenic cell death (ICD), while the late increase of ecto-CRT is related to the removal of apoptotic cells by macrophages.

Binding of natural cytotoxicity receptor NKp46 to sulfate- and α2,3-NeuAc-containing glycans and its mutagenesis.

Natural cytotoxicity receptor 1 (NCR1, NKp46) binds to heparin and heparan sulfate; however, other natural ligands for NKp46 have yet to be elucidated. Using the recombinant extracellular region (coding for AA 22-258) of NKp46 tagged with 6× His (NKp46-H6), and mutants K136Q, R139Q, H142Q, R145Q, and K149Q, we determined their binding affinities to sulfate- and NeuAc-containing glycans-coated plates. NKp46-H6 directly bound to plates coated with heparin- and heparan sulfate-conjugated bovine serum albumin with K(d) values of 770 and 850 nM, respectively. The binding of NKp46-H6 to heparin-BSA was suppressed by soluble heparin, herparan sulfate, fucoidan, λ-carrageenan, and dextran sulfate, but not by 2-O-, 6-O-, and N-desulfated heparin. NKp46-H6 also bound to multimeric sialyl Lewis X expressing transferrin secreted by human hepatoma HepG2 cells (HepTF) with a K(d) value of 530 nM, but not to desialylated HepTF, commercially available TF, or 1-acid glycoprotein. Moreover, mutants R139Q, R145Q, and K149Q had significantly reduced binding to these sulfate-containing glycans, and K136Q and K149Q to HepTF, indicating that NKp46 binds to sulfate- and 2,3-NeuAc-containing glycans mainly via ionic interactions. However, the binding sites of NKp46 were different.

Decreases in CD31 and CD47 levels on the cell surface during etoposide-induced Jurkat cell apoptosis.

Engulfment of apoptotic cells is regulated by 'eat me' and 'don't eat me' signals on the cell surface. Alterations to the 'eat me' signals have been well described; however, very little is known about the 'don't eat me' signals on the cell surface during apoptosis. In the present study, apoptosis of Jurkat cells was induced by treatment with topoisomerase II inhibitor etoposide, and then the CD31 and CD47 levels on the apoptotic cell surface and in microparticles were estimated by flow cytometry and immunoblotting methods in the presence of caspase, metalloproteinase, and Rho-associated coiled-coil containing protein kinase 1 (ROCK1) inhibitors. The CD31 and CD47 levels on the cell surface of apoptotic Jurkat cells had decreased after treatment with etoposide. These decreases in CD31 and CD47 levels on the apoptotic cell surface were almost completely suppressed by the caspase 3 inhibitor, Ac-DEVD-CHO, and partially suppressed by caspase 8 (Ac-IETD-CHO) and caspase 9 (Ac-LEHE-CHO) inhibitors but not by the metalloproteinase inhibitors GM6001 and TAPI-0. Microparticle counts in culture supernatants were higher during etoposide-induced apoptosis. The ROCK1 inhibitor, Y27632, suppressed blebbing formation and microparticle release. Moreover, flow cytometry and immunoblotting revealed CD31 and CD47 in the microparticles. These results indicate that CD31 and CD47 were released by the apoptotic Jurkat cells into the culture supernatant in microparticles, but not in soluble forms, resulting in decreased levels on the apoptotic cell surface.

Binding affinities of NKG2D and CD94 to sialyl Lewis X-expressing N-glycans and heparin.

Lectin-like receptors natural killer group 2D (NKG2D) and CD94 on natural killer (NK) cells bind to α2,3-NeuAc-containing N-glycans and heparin/heparan sulfate (HS). Using recombinant glutathione S-transferase-fused extracellular lectin-like domains of NKG2D (rGST-NKG2Dlec) and CD94 (rGST-CD94lec), we evaluated their binding affinities (K(d)) to high sialyl Lewis X (sLeX)-expressing transferrin secreted by HepG2 cells (HepTf) and heparin-conjugated bovine serum albumin (Heparin-BSA), using quartz crystal microbalance (QCM) and enzyme immunoassay (EIA) microplate methods. K(d) values obtained by linear reciprocal plots revealed good coincidence between the two methods. K(d) values of rGST-NKG2Dlec obtained by QCM and EIA, respectively, were 1.19 and 1.11 µM for heparin-BSA >0.30 and 0.20 µM for HepTf, while those of rGST-CD94lec were 1.31 and 1.45 µM for HepTf >0.37 and 0.36 µM for heparin-BSA. These results suggested that these glycans can interact with NKG2D and CD94 to modulate NK cell-dependent cytotoxicity.

Natural killer group 2A (NKG2A) and natural killer group 2C (NKG2C) bind to sulfated glycans and α2,3-NeuAc-containing glycoproteins.

Killer lectin-like receptors on natural killer (NK) cells mediate cytotoxicity through glycans on target cells. We prepared recombinant glutathione S-transferase-fused extracellular lectin-like domains (AA 94-231) of natural killer group 2A (NKG2A) (rGST-NKG2A) and NKG2C (rGST-NKG2C) and determined the binding of these receptors to plates coated with heparin-conjugated bovine serum albumin (heparin-BSA) and glycoproteins. rGST-NKG2A and rGST-NKG2C directly bound to heparin-BSA with K(d) values of 20 and 40 nM, respectively. Binding of rGST-NKG2A and rGST-NKG2C to heparin-BSA was suppressed in the presence of soluble heparin, heparan sulfate, fucoidan, λ-carrageenan, and dextran sulfate. 2-O-Sulfate residues in heparin were essential for the binding of rGST-NKG2A and rGST-NKG2C. Moreover, rGST-NKG2A and rGST-NKG2C bound to multimeric sialyl Lewis X expressing transferrin secreted by HepG2 cells with K(d) values of 80 and 114 nM, respectively. This is the first report showing that NKG2A and NKG2C bind to heparin and α2,3-NeuAc-containing glycoproteins.

N-terminal region of RecQ4 inhibits non-homologous end joining and chromatin association of the Ku heterodimer in Xenopus egg extracts.

RecQ4, a member of the RecQ helicase family, is required for the maintenance of genome integrity. RecQ4 has been shown to promote the following two DNA double-strand break (DSB) repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). However, its molecular function has not been fully elucidated. In the present study, we aimed to investigate the role of RecQ4 in NHEJ using Xenopus egg extracts. The N-terminal 598 amino acid region of Xenopus RecQ4 (N598), which lacks a central helicase domain and a downstream C-terminal region, was added to the extracts and its effect on the joining of DNA ends was analyzed. We found that N598 inhibited the joining of linearized DNA ends in the extracts. In addition, N598 inhibited DSB-induced chromatin binding of Ku70, which is essential for NHEJ, while the DSB-induced chromatin binding of the HR-associated proteins, replication protein A (RPA) and Rad51, increased upon the addition of N598. These results suggest that RecQ4 possibly influences the choice of the DSB repair pathway by influencing the association of the Ku heterodimer with the DNA ends.

Altered expression of glycoproteins on the cell surface of Jurkat cells during etoposide-induced apoptosis: shedding and intracellular translocation of glycoproteins.

BACKGROUND: The glycoproteins on the cell surface are altered during apoptosis and play an important role in phagocytic clearance of apoptotic cells. METHODS: We classified Jurkat cells treated with etoposide as viable and early apoptotic cells, late apoptotic cells or secondary necrotic cells based on propidium iodide staining and scattered grams and estimated the expression levels of glycoproteins on the cell surface. RESULTS: The cell surface expression levels of intercellular adhesion molecules (ICAM)-2 and -3 on the apoptotic cells were markedly lower, while those of calnexin, calreticulin, and lysosome-associated membrane proteins (LAMP)-1 and -2 were significantly higher compared to non-apoptotic cells. These decreases in ICAM-2 and -3 on the apoptotic cell surface were reduced in the presence of metalloproteinase inhibitors and caspase inhibitors, respectively. Confocal microscopic analysis revealed that calnexin and calreticulin were assembled around fragmented nuclei of blebbed apoptotic cells. CONCLUSIONS: These results suggest that alteration of glycoproteins on the cell surface during apoptosis is associated with shedding and intracellular translocation of glycoproteins.

Prolonged high glucose suppresses phorbol 12-myristate 13-acetate and ionomycin-induced interleukin-2 mRNA expression in Jurkat cells.

BACKGROUND: The disturbance of immunological responses is a complication of diabetes mellitus. METHODS AND RESULTS: We cultured Jurkat cells in 11.1 (normal) and 22.2 mmol/l (high) glucose for 12 weeks and stimulated them with 10 nmol/l phorbol 12-myristate 13-acetate (PMA) and 500 nmol/l ionomycin. RT-PCR revealed that induced interleukin (IL)-2 mRNA expression levels were suppressed in high glucose cultures compared to those in normal glucose. Promoter activities of IL-2, nuclear factor of activated T cells (NFAT), and activator protein-1 (AP-1), after 6 h stimulation with PMA and ionomycin, gradually decreased in high glucose cultures to approximately 20% of those in normal glucose at 12 weeks. The prolonged culture in high glucose increased inducible cAMP early repressor (ICER) II mRNA and protein levels, and overexpression of ICER II dose-dependently suppressed promoter activities of IL-2, NFAT, and AP-1. Moreover, ICER II mRNA expression was transiently induced by stimulation with PMA and ionomycin in normal glucose cultures; however, with high glucose, the induction disappeared. CONCLUSION: These results indicate that ICER II protein accumulates during prolonged culture in high glucose and suppresses IL-2 mRNA expression in Jurkat cells.

Glycated human serum albumin enhances macrophage inflammatory protein-1beta mRNA expression through protein kinase C-delta and NADPH oxidase in macrophage-like differentiated U937 cells.

BACKGROUND: In a previous report (Higai K et al., Biol Pharm Bull, 2007), glycated human serum albumin (Glc-HSA) was found to induce interleukin-8 (IL-8) mRNA expression in human monocyte-derived U937 cells through a reactive oxygen species (ROS)-dependent pathway; however, Glc-HSA signaling has not been elucidated in macrophages. METHODS: U937 cells were differentiated by treatment with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) for 2 days and the macrophage-like differentiated U937 (differentiated U937) cells were stimulated with Glc-HSA and glycolaldehyde dimer-modified HSA (GA-HSA) in the presence of various signaling inhibitors. Macrophage inflammatory protein-1beta (MIP-1beta) mRNA expression was determined by real-time PCR. Intracellular ROS generation was estimated by confocal laser microscopy. RESULTS: Glc-HSA and GA-HSA markedly enhanced MIP-1beta mRNA expression in differentiated U937 cells. Enhanced MIP-1beta mRNA expression was completely suppressed by the ROS scavenger N-acetyl-l-cysteine, the NADPH oxidase inhibitors diphenylene iodonium and apocynin, and the protein kinase C (PKC)-delta inhibitor rottlerin. Furthermore, ROS generation was suppressed completely by rottlerin but not by the PKC-gamma inhibitor Ro318425 or the PKC-alpha, -beta1 and -micro inhibitor Go6976. CONCLUSION: Glc-HSA and GA-HSA enhance MIP-1beta mRNA expression in differentiated U937 cells through PKC-delta-dependent activation of NADPH oxidase.

NKG2D and CD94 bind to multimeric alpha2,3-linked N-acetylneuraminic acid.

Killer lectin-like receptors on natural killer cells mediate cytotoxicity through glycans on target cells including the sialyl Lewis X antigen (sLeX). We investigated whether NK group 2D (NKG2D) and CD94 can bind to sialylated N-linked glycans, using recombinant glutathione S-transferase-fused extracellular lectin-like domains of NKG2D (rNKG2Dlec) and CD94 (rCD94lec). Both rNKG2Dlec and rCD94lec bound to plates coated with high-sLeX-expressing transferrin secreted by HepG2 cells (HepTF). The binding of rNKG2Dlec and rCD94lec to HepTF was markedly suppressed by treatment of HepTF with neuraminidase and in the presence of N-acetylneuraminic acid. Moreover, rNKG2Dlec and rCD94lec bound to alpha2,3-sialylated human alpha(1)-acid glycoprotein (AGP) but not to alpha2,6-sialylated AGP. Mutagenesis revealed that (152)Y of NKG2D and (144)F and (160)N of CD94 were critical for HepTF binding. This is the first report that NKG2D and CD94 bind to alpha2,3-sialylated but not to alpha2,6-sialylated multi-antennary N-glycans.

NKG2D and CD94 bind to heparin and sulfate-containing polysaccharides.

Killer lectin-like receptors NKG2D and CD94 on natural killer cells trigger cytotoxicity through binding of glycans on target cells including sialyl Lewis X antigen. We previously reported that NKG2D and CD94 recognize alpha2,3-linked NeuAc on multi-antennary N-glycans. Here we further investigated polysaccharide binding by these receptors, using glutathione-S-transferase-fused extracellular domains of NKG2D AA 73-216 (rNKG2Dlec) and CD94 AA 68-179 (rCD94lec). We found that rNKG2Dlec and rCD94lec bind in a dose-dependent manner to plates coated with heparin-conjugated bovine serum albumin (heparin-BSA). Binding to heparin-BSA was suppressed by soluble sulfate-containing polysaccharides, but minimally impacted by 2-O-, 6-O-, and 2-N-desulfated heparin. Mutagenesis revealed that (152)Y and (199)Y of NKG2D and (144)F, (160)N, and (166)C of CD94 were critical for binding to heparin-BSA. The present manuscript provides the first evidence that NKG2D and CD94 bind to heparin and sulfate-containing polysaccharides.

Immobilized alpha2,6-linked sialic acid suppresses caspase-3 activation during anti-IgM antibody-induced apoptosis in Ramos cells.

In Ramos cells, a human Burkitt's lymphoma cell line, stimulation of the B cell antigen receptor with anti-IgM antibody (Ab) induces apoptosis as indicated by a decrease in cell viability and an increase in DNA fragmentation and cell surface exposure of phosphatidylserine. Furthermore, these changes are suppressed by incubating the cells in alpha(1)-acid glycoprotein (AGP)-coated tissue culture plates. Here, we found that, during Anti-IgM Ab-induced apoptosis in Ramos cells, caspase-3 is activated downstream of caspase-8 and the mitochondrial pathway is activated, as indicated by a loss of mitochondrial membrane potential, an increase in the release of cytochrome c to the cytoplasm, and enhanced Bax expression. Anti-IgM Ab-induced apoptosis of neuraminidase-treated Ramos cells was suppressed by incubating the cells on plates coated with AGP, which contains a high concentration of alpha2,6-linked sialic acid. The incubation on plates coated with AGP also suppressed anti-IgM Ab-stimulated caspase-3 activity and increased the level of X-linked inhibitor of apoptosis protein (XIAP), but it did not affect caspase-8 activity, the mitochondrial membrane potential, cytochrome c release, or Bax expression. The results indicate that the interaction of Ramos cells with immobilized alpha2,6-linked sialic acid enhances XIAP expression, directly or indirectly suppressing caspase-3 activity and inhibiting anti-IgM Ab-induced apoptosis.

Glycosylation of site-specific glycans of alpha1-acid glycoprotein and alterations in acute and chronic inflammation.

BACKGROUND: alpha(1)-Acid glycoprotein (AGP), an acute phase reactant, is extensively glycosylated at five Asn-linked glycosylation sites. In a number of pathophysiological states, including inflammation, rheumatoid arthritis, and cancer, alterations of Asn-linked glycans (N-glycans) have been reported. We investigated alteration of N-glycans at each of glycosylation sites of AGP in the sera of patients with acute and chronic inflammation. METHODS: AGP purified from sera was digested with Glu-C and the liberated glycopeptides were isolated by reverse phase HPLC. N-glycans released with peptide N-glycosidase F and followed by neuraminidase treatment were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. RESULTS: Site-specific differences in branching structures were observed among N-glycosylation sites 1, 3, 4 and 5. Within the sera of patients with acute inflammation, increases in bi-antennary and decreases in tri- and tetra-antennary structures were observed, as well as increases in alpha1,3-fucosylation, at most glycosylation sites. In the sera of patients with chronic inflammation, increased rates of tri-antennary alpha1,3-fucosylation at sites 3 and 4 and tetra-antennary alpha1,3-fucosylation at sites 3, 4 and 5 were detected. Although there were no significant differences between acute and chronic sera in site directed branching structures, significant differences of alpha1,3-fucosylation were detected in tri-antennary at sites 2, 4 and 5 and in tetra-antennary at sites 3 and 4. CONCLUSION: Little variation in the N-glycan composition of the glycosylation sites of AGP was observed among healthy individuals, while the sera of patients with acute inflammation demonstrated increased numbers of bi-antennary and alpha1,3-fucosylated N-glycan structures at each glycosylation site.

Interleukin-1beta induces sialyl Lewis X on hepatocellular carcinoma HuH-7 cells via enhanced expression of ST3Gal IV and FUT VI gene.

We previously demonstrated that human hepatocellular carcinoma-derived HuH-7 cells stimulated with interleukin-1beta (IL-1beta) produce alpha(1)-acid glycoprotein (AGP) with increased amounts of sialyl Lewis X (sLeX) antigen, although the mechanism remained obscure. Here, we report our investigation of the mechanism. sLeX expression on HuH-7 cells was induced 2.5 times more after 48 h stimulation with 100 U/mL IL-1 beta compared with control, as indicated by anti-sLeX antibody binding. Furthermore, expression of 2,3-sialylated N-acetyllactosamine increased gradually up to 48 h after IL-1 beta stimulation; this preceded the increase in sLeX expression. Increases in alpha 2,3-sialyltransferase activity also preceded increases in alpha1,3-fucosyltransferase activity. Furthermore, mRNA levels of ST3Gal IV, FUT IV and VI in HuH-7 cells stimulated with IL- 1beta were increased at 2-4 h, while increases in FUT VI mRNA level occurred gradually after 24 h. IL-1 beta-induced sLeX expression on HuH-7 cells was suppressed by transfection of gene-specific small interference RNAs against FUT VI and ST3Gal IV but not against FUT IV and ST3Gal III. These data results that IL-1 beta induces expression of sLeX on HuH-7 cells by enhanced expression of FUT VI and ST3Gal IV gene.